EP3066107B1 - Chromogenes substrat für beta-d-glucuronidase und dessen verwendung in mikrobennachweis - Google Patents

Chromogenes substrat für beta-d-glucuronidase und dessen verwendung in mikrobennachweis Download PDF

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EP3066107B1
EP3066107B1 EP14793275.0A EP14793275A EP3066107B1 EP 3066107 B1 EP3066107 B1 EP 3066107B1 EP 14793275 A EP14793275 A EP 14793275A EP 3066107 B1 EP3066107 B1 EP 3066107B1
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group
compound
independently selected
metal cation
formula
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EP3066107A1 (de
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Hayley Jane TURNER
Michael Burton
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Glycosynth Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
    • G01N2333/942—Hydrolases (3) acting on glycosyl compounds (3.2) acting on beta-1, 4-glucosidic bonds, e.g. cellulase

Definitions

  • This invention relates to chromogenic enzyme substrates used to detect E . coli and other ⁇ -D-glucuronidase-producing microorganisms in liquid growth media.
  • C-EC Agar Biolife Italiana Srl, Milan, Italy
  • fluorogenic MUG 4-methylumbelliferyl ⁇ -D-glucuronide
  • chromogenic X-Gal 5-bromo-4-chloro-3-indolyl ⁇ -D-galactopyranoside
  • Coliscan® (Micrology LLC, Goshen, USA), Chromocult ® Coliform agar (Merck, Darmstadt, Germany) and Chromogenic Coliform Agar (Biolife Italiana Srl, Milan, Italy) adopt a dual chromogenic system with the substrates X- ⁇ -D-glucuronide (5-bromo-4-chloro-3-indolyl ⁇ -D-glucuronide) [dark-blue to violet colour with E. coli ] and 6-chloro-3-indolyl ⁇ -D-galactopyranoside [red colonies with other coliforms]. It will be noted that all of the above-mentioned media contain indoxyl substrates.
  • Indoxyl substrates work very well in solid media such as agar plates, but they are much less suited to liquid media, mainly because the indigo chromogen they produce is very insoluble. In the few examples of liquid media for detecting E. coli and total coliforms which do contain an indoxyl substrate, it is a galactoside for the visualisation of total coliforms.
  • Fluorocult® LMX broth and Readycult® Coliforms 100 Merck, Darmstadt, Germany
  • LSB X-Gal MUG Biolife Italiana Srl, Milan, Italy
  • Resorufin- ⁇ -D-glucuronide is another extremely expensive substrate, as is fluorescein-di- ⁇ -D-glucuronide.
  • Methyl esters of glucuronides have been used as substrates for ⁇ -D-glucuronidase activity.
  • Fluorescein-di- ⁇ -D-glucuronide dimethyl ester is a product of Marker Gene Technologies, Inc. (Eugene, Oregon, USA) [catalogue number M0969] and has been advertised as a ⁇ -D-glucuronidase substrate since 2004 [ Marker Gene Technologies Newsletter, Vol. 4, Nos. 9 and 10, 2004 ].
  • the manufacturers state that this substrate has enhanced cell-permeation properties in plants.
  • glucuronide substrate of limited commercial availability that has been used to detect E. coli , namely 8-hydroxyquinoline- ⁇ -D-glucuronide.
  • this substrate is cleaved the aglycone forms a highly insoluble intense black chelate with iron compounds [ A.L. James and P. Yeoman, explicatbl. Bakteriol. Mikrobiol. Hyg. A, 267, 188, (1987 )].
  • the substrate has been demonstrated in an agar plate medium [ R.D. Reinders et al, Lett. Appl. Microbiol., 30, 411-414, (2000 )]
  • toxicity of the aglycone to Gram-positive microbes has been observed [ J.D. Perry et al, J. Appl. Microbiol., 102, 410-415, (2007 )].
  • the substrate is not suitable for liquid media.
  • Enzyme substrates based on catechol have been described in EP1438423 .
  • this invention is restricted to chromogenic substrates giving a non-diffusible endpoint on solid media such as agar plates.
  • Catechol- ⁇ -D-glucuronide is not disclosed in EP1438423 .
  • catechol- ⁇ -D-glucuronide has been mentioned occasionally in the scientific literature as a putative metabolic by-product from glucuronidation of either catechol or phenol, both the complete chemical synthesis and the characterisation of this glucuronide have yet to be described.
  • Luukkanen L et al. "Enzyme-assisted synthesis and structural characterization of nitrocatechol glucuronides", Bioconjugate Chemistry, ACS, Washington DC, US, Vol. 10, 1 January 1999 (1999-01-01), pages 150-154 disclose six ⁇ -glucuronyl compounds derived from nitrocatechols by an enzymatic synthesis. The compounds are stated to be of potential use for enzyme kinetic studies in vitro and for use as reference standards required in pharmacokinetic investigations and metabolism studies of glucuronides.
  • This invention provides a method, compound, composition and synthesis for the detection of E. coli and other ⁇ -D-glucuronidase-producing microorganisms in liquid growth media.
  • the invention provides a method of detecting ⁇ -D-glucuronidase activity in a liquid medium comprising the steps of:
  • the compound of the formula I is a substrate for -D-glucuronidase, and is a glucuronide of an aromatic compound having two vicinal OH substituents in an aryl ring.
  • the carboxylic acid group of the glucuronic unit may be in the form of the free acid (X is H) or may be esterified (X is alkyl) or may be in the form of a salt of an inorganic or organic base. In the latter case the carboxylic group is in the form of an anion, while X is a cation and the ions are electrostatically bound.
  • Non-metal cations encompass inorganic cations, such as NH + 4 , and organic cations.
  • An organic base is an ammonium compound e.g.
  • An inorganic base is ammonium hydroxide or an oxide or hydroxide of an alkali metal, an alkaline earth metal or another metal M n+ 1/n where n is 1, 2 or 3.
  • the compound of the formula I is a compound of the general formula II wherein X 2 is independently selected from the group consisting of H, CH 3 , C 2 -C 6 alkyl, a metal cation and a non-metal cation; wherein Y 2 is independently selected from the group consisting of H, a metal cation and a non-metal cation; wherein R 21 is H or OH; wherein R 24 is H or NO 2 ; and wherein R 22 and R 23 are H or one of R 22 and R 23 is OH and the other is H, or R 22 and R 23 together form the group VI I-.
  • the composition may itself be a growth medium or may be a premix for adding to a growth medium, or a concentrate from which a growth medium, usually a liquid growth medium, may be formed, e.g. by dilution.
  • Carbon sources may include tryptone, peptone, casein and sugars, preferably lactose and glucose.
  • Nitrogen sources may include amino acids, tryptone, peptone, casein extract, and ammonium sulphate.
  • a microbial growth medium may contain antibacterial or antifungal compounds to aid in selecting and amplifying the microbes of interest.
  • the ⁇ -D-glucuronide substrate is a glucuronide derivative of a vicinal aromatic diol.
  • the free diol moiety is capable of chelating the metal of the metal compound, which is preferably an iron compound.
  • the metal-diol complex is coloured and thus suitable to report the presence of ⁇ -D-glucuronidase activity.
  • a ⁇ -D-glucuronidase Prior to cleavage by a ⁇ -D-glucuronidase, one of the hydroxyl oxygen atoms is bound to the glucuronide moiety via a glycosidic bond, which prevents chelation and hence colour formation. Thus, it is only in the presence of a ⁇ -D-glucuronidase, which cleaves the glycosidic bond that a colour develops in the method of claim 1.
  • Acyl and acyloxy groups R 2 and R 3 and R 5 to R 8 usually are alkanoyl and alkanoyloxy, respectively preferably lower alkanoyl and lower alkanoyloxy, respectively but may alternatively be benzoyl and benzoyloxy, respectively.
  • R 24 is H. In an alternative embodiment R 24 is nitro (NO 2 ). In both these embodiments R 22 and R 23 may be said linked group VII.
  • the liquid medium composition of the fourth aspect may comprise agar or any other gelling agent in order to increase the viscosity of the medium to provide ease of handling.
  • the leaving group L is selected from conventional leaving groups. Examples are halogen, preferably Br, and acyloxy, preferably acetoxy or trichloroacetimidyloxy.
  • NMR spectra were recorded on a 270 MHz Joel NMR spectrometer (at 270 MHz for 1H and 68 MHz for 13C) or NMR spectra were recorded on a 400MHz Joel NMR spectrometer (at 400 MHz for 1H and 100 MHz for 13C). All chemical shifts are quoted in ppm relative to TMS. Optical rotations were measured on an Optical Activity AA10 polarimeter. Melting points were determined with an Electrothermal AI9200 apparatus and are uncorrected. All melting points are quoted to the nearest 0.5°C. High Resolution Mass Spectroscopy (HRMS) data were obtained using the EPSRC mass spectrometry service centre (Swansea, UK).
  • HRMS High Resolution Mass Spectroscopy
  • the iron compound can be either iron(II) (i.e. ferrous) or iron(III) (i.e.
  • Rose- ⁇ -D-glucuronide CHA salt (Glycosynth Ltd, Warrington, UK) (20 mg) was dissolved in NMP (200 ⁇ L). These solutions were then added to Columbia agar (Oxoid, Basingstoke, UK) (100 mL) and inoculated with bacterial suspensions made up to 0.5 McFarland standard (1 ⁇ L). The strains of E.coli used are listed in table 2. The plates and broths were incubated at 37°C for 18 hours in air. The green colonies seen on the CPS ID 3 media were indicative of ⁇ -D-glucosidase activity. Table 2.

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Claims (19)

  1. Verfahren zum Nachweisen von β-D-Glucuronidaseaktivität in einem flüssigen Medium, die folgenden Schritte umfassend:
    a) Inberührungbringen einer Metallverbindung und eines β-D-Glucuronidasesubstrats mit der folgenden Formel I mit einer Substanz, von der vermutet wird, dass sie eine Glucuronidase enthält oder erzeugt:
    Figure imgb0021
     wobei X unabhängig ausgewählt ist aus der Gruppe bestehend aus H, CH3, C2-C6-Alkyl und einem Metallkation und einem Nichtmetallkation;
    wobei Y unabhängig ausgewählt ist aus der Gruppe bestehend aus H, einem Metallkation und einem Nichtmetallkation;
    wobei entweder
    i) R1 H oder OH ist und R4 H oder NO2 ist; oder
    ii) R1 und R4 unabhängig ausgewählt sind aus der Gruppe bestehend aus H, C1-C8-Alkyl, C1-C8-Alkoxy, C1-C8-Hydroxyalkyl, Halogen, Nitro, C2-C24-Acyl, C2-C24-Acyloxy, C7-C24-Aralkyl, C6-C24-Aryl, Sulfonyl und Amido; und
    wobei R2 und R3 unabhängig ausgewählt sind aus der Gruppe bestehend aus H, OH, C1-C8-Alkyl, C1-C8-Alkoxy, C1-C8-Hydroxyalkyl, Halogen, Nitro, C2-C24-Acyl, C2-C24-Acyloxy, C7-C24-Aralkyl, C6-C24-Aryl, Sulfonyl und Amido unter der Voraussetzung, dass R2 und R3 nicht beide OH darstellen, oder R2 und R3 gemeinsam die Gruppe ausbilden, die durch die Formel VI dargestellt wird:

            -CR5=CR6-CR7=CR8-     VI;

    wobei R5-R8 unabhängig ausgewählt sind aus der Gruppe bestehend aus H, C1-C8-Alkyl, C1-C8-Alkoxy, C1-C8-Hydroxyalkyl, Halogen, Nitro, C2-C24-Acyl, C2-C24-Acyloxy, C7-C24-Aralkyl, C6-C24-Aryl, Hydroxyl, Sulfonyl und Amido unter der Voraussetzung, dass in der Gruppe VI jegliche benachbarte Gruppen R5 bis R8 nicht beide OH sind;
    sodass ein Produkt der Substratspaltung in der Lage ist, eine Chelatbildung der Metallverbindung zu bewirken, um somit eine farbige Verbindung auszubilden; und
    b) Nachweisen des Vorliegens der farbigen Verbindung.
  2. Verfahren nach Anspruch 1, wobei das Substrat durch die folgende Formel II dargestellt wird:
    Figure imgb0022
     wobei X2 unabhängig ausgewählt ist aus der Gruppe bestehend aus H, CH3, C2-C6-Alkyl, einem Metallkation und einem Nichtmetallkation;
    wobei Y2 unabhängig ausgewählt ist aus der Gruppe bestehend aus H, einem Metallkation und einem Nichtmetallkation;
    wobei R21 H oder OH ist;
    wobei R24 H oder NO2 ist; und
    wobei R22 und R23 H sind oder einer aus R22 und R23 H ist und der andere OH ist oder R22 und R23 gemeinsam die Gruppe VII ausbilden:

            -CR25=CR26-CR27=CR28-     VII;

    wobei R25 bis R28unabhängig ausgewählt sind aus H, OH, C1-C8-Alkyl, C1-C8-Alkoxy, C1-C8-Hydroxyalkyl, Halogen, Nitro, C2-C24-Acyl, C2-C24-Acyloxy, C7-C24-Aralkyl, C6-C24-Aryl, Sulfonyl und Amido, unter der Voraussetzung, dass jegliche benachbarte Gruppen R25-R28 nicht beide OH sind.
  3. Verfahren nach einem der vorhergehenden Ansprüche, wobei die Substanz, von der angenommen wird, dass sie eine β-D-Glucuronidase enthält, bakteriell ist, vorzugsweise wobei Schritt a) des Verfahrens das Inkubieren des flüssigen Mediums in der Gegenwart des Substrats, der Metallverbindung und der Substanz beinhaltet, vorzugsweise
    wobei die Bakterien Escherichia coli sind oder aus den Gattungen Shigella oder Salmonella stammen.
  4. Verfahren nach einem der vorhergehenden Ansprüche, wobei das flüssige Medium wenigstens ein weiteres Enzymsubstrat enthält, das für ein von β-D-Glucuronidase verschiedenes bakterielles Enzym vorgesehen ist, vorzugsweise wobei das andere Enzymsubstrat ein Substrat für β-D-Galactosidase ist, vorzugsweise ein chromogenes Enzymsubstrat, und wobei das Verfahren den Schritt des Nachweisens des farbigen Produkts von β-D-Galactosidasespaltung enthält, stärker bevorzugt wobei das andere Enzymsubstrat o-Nitrophenyl-β-D-galactopyranosid ist.
  5. Verbindung, dargestellt durch die Formel II:
    Figure imgb0023
     wobei X2 unabhängig ausgewählt ist aus der Gruppe bestehend aus H, CH3, C2-C6-Alkyl, einem Metallkation und einem Nichtmetallkation;
    wobei Y2 unabhängig ausgewählt ist aus der Gruppe bestehend aus H, einem Metallkation und einem Nichtmetallkation;
    wobei R21 H ist;
    wobei R24 H oder NO2 ist; und
    wobei R22 und R23 H sind oder einer OH ist und der andere H ist oder R22 und R23 gemeinsam die Gruppe VII ausbilden:

            -CR25=CR26-CR27=CR28-     VII;

    wobei R25 bis R28unabhängig ausgewählt sind aus H, OH, C1-C8-Alkyl, C1-C8-Alkoxy, C1-C8-Hydroxyalkyl, Halogen, Nitro, C2-C24-Acyl, C2-C24-Acyloxy, C7-C24-Aralkyl, C6-C24-Aryl, Sulfonyl und Amido, unter der Voraussetzung, dass jegliche benachbarte Gruppen R25-R28 nicht beide OH sind.
  6. Verbindung nach Anspruch 5, wobei X2 H, CH3, NH4 + oder ein Kation, ausgewählt aus der Gruppe bestehend aus primärem Ammonium, sekundärem Ammonium, tertiärem Ammonium, quartärem Ammonium, Li+, Na+, K+, Cs+, Ca1/2 2+, Mg1/2 2+, Rb+, Sr1/2 2+ und Ba1/2 2+ ist, vorzugsweise wobei X2 ausgewählt ist aus den folgenden Optionen:
    a) wobei X2 ein Ammoniumkation ist, ausgewählt aus der Gruppe bestehend aus Ammonium (NH4 +), Cyclohexylammonium, 2-Methyl-1,3-propandiol-2-ammonium, Phenylammonium, 3-Methylphenylammonium, 4-Methylphenylammonium und 2-Methylphenylammonium;
    b) wobei X2 ausgewählt ist aus der Gruppe bestehend aus H, NH4 +, Cyclohexylammonium, Li+, Na+ und K+; und
    c) wobei X2 CH3 ist.
  7. Verbindung nach Anspruch 5 oder 6, wobei R21 bis R24 jeweils H sind, ausgewählt aus den folgenden Optionen:
    a) wobei R21 bis R24 jeweils H sind;
    b) wobei R21 und R24 jeweils H sind und R22 und R23 gemeinsam mit den Atomen, an die sie gebunden sind, den fusionierten Phenylring ausbilden, vorzugsweise wobei jeder aus R25 bis R28 H ist; und
    c) wobei R21 OH ist und R22 bis R24 jeweils H sind.
  8. Verbindung nach einem der Ansprüche 5 bis 7, wobei Y2 H ist.
  9. Verbindung nach Anspruch 5, wobei X2 und Y2 beide entweder Natrium oder Kalium sind.
  10. Verbindung nach Anspruch 5 oder 6, wobei R21 OH ist.
  11. Zusammensetzung, Folgendes umfassend:
    a) eine Verbindung, dargestellt durch die Formel I:
    Figure imgb0024
     wobei X unabhängig ausgewählt ist aus der Gruppe bestehend aus H, CH3, C2-C6-Alkyl und einem Metallkation und einem Nichtmetallkation;
    wobei Y unabhängig ausgewählt ist aus der Gruppe bestehend aus H, einem Metallkation und einem Nichtmetallkation;
    wobei entweder
    i) R1 H oder OH ist; und R4 H oder NO2 ist; oder
    ii) R1 und R4 unabhängig ausgewählt sind aus der Gruppe bestehend aus H, C1-C8-Alkyl, C1-C8-Alkoxy, C1-C8-Hydroxyalkyl, Halogen, Nitro, C2-C24-Acyl, C2-C24-Acyloxy, C7-C24-Aralkyl, C6-C24-Aryl, Sulfonyl und Amido; und
    wobei R2 und R3 unabhängig ausgewählt sind aus der Gruppe bestehend aus H, OH, C1-C8-Alkyl, C1-C8-Alkoxy, C1-C8-Hydroxyalkyl, Halogen, Nitro, C2-C24-Acyl, C2-C24-Acyloxy, C7-C24-Aralkyl, C6-C24-Aryl, Sulfonyl und Amido unter der Voraussetzung, dass R2 und R3 nicht beide OH darstellen, oder R2 und R3 gemeinsam eine Gruppe mit der Formel VI ausbilden:

            -CR5=CR6-CR7=CR8-     VI;

    wobei R5-R8 unabhängig ausgewählt sind aus der Gruppe bestehend aus H, OH, C1-C8-Alkyl, C1-C8-Alkoxy, C1-C8-Hydroxyalkyl, Halogen, Nitro, C2-C24-Acyl, C2-C24-Acyloxy, C7-C24-Aralkyl, C6-C24-Aryl, Sulfonyl und Amido, unter der Voraussetzung, dass jegliche benachbarte Gruppen R5-R8 nicht beide OH sind; und
    b) eine Metallverbindung.
  12. Verbindung nach Anspruch 11, bei der es sich um ein Konzentrat handelt, das nach Verdünnung ein mikrobielles Wachstumsmedium ausbildet.
  13. Flüssige Mediumzusammensetzung, die geeignet ist zum Züchten von Mikroorganismen, Folgendes umfassend:
    a) ein β-D-Glucuronid mit der folgenden Formel I:
    Figure imgb0025
     wobei X unabhängig ausgewählt ist aus der Gruppe bestehend aus H, CH3, C2-C6-Alkyl und einem Metallkation und einem Nichtmetallkation;
    wobei Y unabhängig ausgewählt ist aus der Gruppe bestehend aus H, einem Metallkation und einem Nichtmetallkation;
    wobei entweder
    i) R1 H oder OH ist und R4 H oder NO2 ist; oder
    ii) R1 und R4 unabhängig ausgewählt sind aus der Gruppe bestehend aus H, C1-C8-Alkyl, C1-C8-Alkoxy, C1-C8-Hydroxyalkyl, Halogen, Nitro, C2-C24-Acyl, C2-C24-Acyloxy, C7-C24-Aralkyl, C6-C24-Aryl, Sulfonyl und Amido; und
    wobei R2 und R3 unabhängig ausgewählt sind aus der Gruppe bestehend aus H, OH, C1-C8-Alkyl, C1-C8-Alkoxy, C1-C8-Hydroxyalkyl, Halogen, Nitro, C2-C24-Acyl, C2-C24-Acyloxy, C7-C24-Aralkyl, C6-C24-Aryl, Sulfonyl und Amido, unter der Voraussetzung, dass R2 und R3 nicht beide OH darstellen, oder dass R2 und R3 gemeinsam mit den Atomen, an die sie gebunden sind, die Gruppe ausbilden, die durch die folgende Formel VI dargestellt wird:

            -CR5=CR6-CR7=CR8-     VI;

    wobei R5-R8 unabhängig ausgewählt sind aus der Gruppe bestehend aus H, OH, C1-C8-Alkyl, C1-C8-Alkoxy, C1-C8-Hydroxyalkyl, Halogen, Nitro, C2-C24-Acyl, C2-C24-Acyloxy, C7-C24-Aralkyl, C6-C24-Aryl, Sulfonyl und Amido unter der Voraussetzung, dass in der Gruppe VI jegliche benachbarte Gruppen R5 bis R8 nicht beide OH sind;
    b) eine Metallverbindung.
  14. Zusammensetzung nach einem der Ansprüche 11 bis 13, umfassend ein Verdickungsmittel, vorzugsweise Agar, zum Beispiel in einer Konzentration von nicht mehr als 0,5 %.
  15. Zusammensetzung nach einem der Ansprüche 11 bis 14, umfassend einen oder mehrere Induktoren von β-D-Glucuronidase, vorzugsweise Glucuronat und/oder ein β-D-Glucuronid, das von dem β-D-Glucuronid mit der Formel I verschieden ist.
  16. Zusammensetzung nach einem der Ansprüche 11 bis 15, wobei das β-D-Glucuronid mit der Formel I eine Verbindung mit der Formel II nach einem der Ansprüche 5 bis 10 ist.
  17. Verfahren nach einem der Ansprüche 1 bis 4 oder Zusammensetzung nach einem der Ansprüche 11 bis 16, wobei die Metallverbindung ein Metallsalz einer organischen oder anorganischen Säure ist, vorzugsweise ein wasserlösliches Salz, stärker bevorzugt wobei die Metallverbindung ein Eisen(II)-Salz, ein Eisen(III)-Salz oder ein gemischtes Eisen(II)/Eisen(III)-Salz ist.
  18. Verfahren für die chemische Synthese einer Verbindung mit der Formel II nach Anspruch 5, die folgende Schritte umfassend:
    a) Konjugieren einer Glucuronsäureverbindung mit der Formel III:
    Figure imgb0026
     mit einer Verbindung mit der Formel IV:
    Figure imgb0027
     um ein Konjugat mit der Formel V auszubilden:
    Figure imgb0028
     wobei L eine Abgangsgruppe ist;
    wobei X1 H, CH3 oder eine Gruppe ist, die in X2 umgewandelt werden kann, wie in der zu synthetisierenden Verbindung mit der Formel II;
    wobei Y1 H ist oder eine Gruppe, die in Y2 umgewandelt werden kann, wie in der zu synthetisierenden Verbindung mit der Formel II;
    wobei jeder Ra eine Hydroxy-Schutzgruppe ist;
    wobei R11 H, OH oder eine Gruppe ist, die in R21 umgewandelt werden kann;
    wobei entweder
    i) R12 H oder eine Gruppe ist, die in R22 umgewandelt werden kann; und
    R13 H, OH oder eine Gruppe ist, die in R23 umgewandelt werden kann, unter der Voraussetzung, dass R22 und R23 nicht beide OH sind; oder
    ii) R12 und R13 gemeinsam eine Gruppe mit der Formel VIII darstellen:

            -CR35=CR36-CR37=CR38     VIII;

    wobei R35-R38 die jeweiligen selben Gruppen wie R25-R28 darstellen oder Gruppen darstellen, die in die jeweiligen Gruppen umgewandelt werden können; und
    wobei R14 H, NO2 oder eine Gruppe ist, die in R24 umgewandelt werden kann;
    b) Entschützen der Gruppen ORa, um OH auszubilden; und optional
    c) Umwandeln der Gruppen X1, Y1, R11, R12, R13, R14, R35, R36, R37 beziehungsweise R38 in X2, Y2, R21, R22, R23, R24, R25, R26, R27 beziehungsweise R28.
  19. Syntheseverfahren nach Anspruch 18, eines der folgenden Merkmale umfassend:
    a) wobei die Konjugation säure- oder basekatalysiert ist;
    b) wobei die Entschützung mit einem Alkali, einer Säure oder durch Hydrogenolyse durchgeführt wird;
    c) wobei L ausgewählt ist aus Halogen, vorzugsweise Br, Acyloxy, vorzugsweise Acetoxy und Trichloracetimidyloxy;
    d) wobei jeder Ra gleich ist und eine Acetyl-, Benzoyl- oder Benzylgruppe ist;
    e) wobei jeder R11 bis R14 H ist und jeder R21 bis R24 H ist;
    f) wobei Y1 H oder ein Metall- oder Nichtmetallkation ist und mit Y2 identisch ist; und
    g) wobei X1 CH3 ist.
EP14793275.0A 2013-11-08 2014-11-03 Chromogenes substrat für beta-d-glucuronidase und dessen verwendung in mikrobennachweis Active EP3066107B1 (de)

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GB0125532D0 (en) 2001-10-24 2001-12-12 Burton Michael Enzyme activity indicators
FR2912423B1 (fr) 2007-02-08 2009-03-20 Biomerieux Sa Milieu de detection et/ou d'identification de bacteries
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